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Title: Development of digital PCR DNA methylation assays for blood plasma-based diagnosis of lung cancer
Author: Brown, Benjamin R. B.
ISNI:       0000 0004 7428 5301
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Lung cancer is the leading cause of cancer-related death and is usually diagnosed at advanced stage leading to poor patient survival. Therefore there is a pressing need for early detection of disease. DNA methylation is an early event in carcinogenesis and a limited number of diagnostic markers have been developed for clinical use. This thesis seeks to address whether the development and application of novel DNA methylation assays can diagnose lung cancer at early stage. Previously identified DNA methylation biomarkers, along with novel targets identified by methylation microarray, were developed in multiplex assay format. Twelve markers were used to screen 417 bronchoalveolar lavage specimens from Liverpool Lung Project (LLP) subjects divided into training and validation sets. The optimal biomarker panel (CDKN2A, RARB and TERT) demonstrated improved clinical sensitivity and specificity (Sensitivity/Specificity: 85.7%/93.8%, AUC: 0.91) compared to previous studies. The optimal methylation algorithm detected more than 60% of stage T1 tumours and 93 cytologically occult lung cancer cases. Eight methylated DNA assays were optimised for use with the newly developed Droplet DigitalTM PCR (ddPCR) platform and a targeted pre-amplification technique, MethPlex enrichment, was developed. I established a comprehensive analytical framework to compare performance of methylation-specific ddPCR and quantitative methylation-specific PCR directly and in combination with MethPlex enrichment. ddPCR demonstrated greater precision and linearity, lower limit of detection (WT1 MethPlex ddPCR LOD95 = 1.86 GE), and discriminated twofold differences in methylated DNA input. MethPlex ddPCR detected DNA methylation more frequently in lung cancer patient plasma than in controls in a retrospective case-control study. Technical methylation controls were consistently and precisely detected at inputs as low as 3 methylated copies. Discriminatory efficiency of marker combinations was inadequate, presumably due to limitations in DNA extraction methodology. DNA methylation biomarker diagnostic performance in bronchoalveolar lavage merits further validation in a prospective trial. MethPlex ddPCR analysis showed great promise, demonstrating highly sensitive DNA methylation detection in technical assessment. It is expected that appropriate DNA extraction procedures and higher cfDNA yields will lead to much improved clinical discriminatory efficiency.
Supervisor: Liloglou, Triantafillos ; Field, John Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral